Assembling Neurospheres: Dynamics of Neural Progenitor/Stem Cell Aggregation Probed Using an Optical Trap

Abstract

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (∼5 s) and most probable minimum distance of approach (4–6 µm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (∼18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and “stemness”. Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion.

Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy

“…[12][13][14][15] It is noteworthy that previous microRaman studies of differentiation have utilized four-fold high levels of incident power. 6,9 As described in other reports from our laboratory, [12][13][14][15] in which the present apparatus was used in the form of a Raman Tweezers setup, we first established the absence of significant photochemistry induced by the 785-nm laser light by recording Raman spectra of a given cell for 10 min at a time, with an interval of 1 min between subsequent measurements. We ensured that all 10 spectra, thus, obtained retained the same Raman features, providing indication that no photoinduced damage was induced in the cell in the course of the experiment.…”

Optically trapping tumor cells to assess differentiation and prognosis of cancers

“…Characterizing the dynamics of the adhesion process and/or the adhesion molecular signature is expected to indicate the differentiation stage of a tumor and its propensity for metastasis. Our trap-based method shows that τ min increases for all untreated malignant cells of both cell lines (mean 27 s and 21 s, respectively) -well over that for normal NSCs (~5 s) [9]. The sensitivity (52-59%) and specificity (72-87.5%) of our cell adhesion assay points to advantages over conventional methods.…”

“…Through a series of measurements (see real-time movie clips) we assessed τ min for different cells (Fig. 2(c), 2(d)) and discovered that they were significantly longer for untreated cells of both cell lines tested (12-44 s, with a mean of 27 s for SK-N-SH cells; 9-36 s, with a mean of 21 s for C6 glioma cells) than that for normal NSCs (~5 s) [9]. ATRA treatment induced dramatic reduction of τ min for all cells from both cell lines, with a minimum time of 4 s and an upper limit of 16 s (Fig.…”

“…Most results were obtained at ~15 mW. By correlating P with the optical force generated [9], we deduced that a trapped cell experienced a force of 10 ± 2 pN, using Stoke's Law to estimate the minimum fluid velocity required to displace it from within the trap. The force is an estimate because drag coefficients are unknown for cell shapes used in our experiments.…”

“…Recently, optical trapping has probed the mechanics and spatio-temporal dynamics of how neural stem cells (NSCs) adhere and aggregate [9] to show that cell-cell adhesion dynamics occur on seconds timescales: two NSCs need to be close to each other for a minimum mean time of ~5 s for adhesion to occur, or before a single NSC adheres to a neurosphere -an aggregate of NSCs. Incorporation of fluid flow methods into the optical trap [9] enabled quantification of a lower limit of ~18 pN as the adhesive force required by proximate NSCs. Prior to the use of a trap, such studies had relied on time-lapse microscopy which accessed only longer timescales (0.5-3 hours) [10].…”